An understanding of the chemical principles for binding specific sites on double helical DNA with oligodeoxyribonucleotides (or their analogs) by triple strand formation would provide a minimum first step in the development of a human therapeutic strategy of genetic targeting. This could ultimately enable precise inactivation of undesirable DNA sequences (such as oncogenes or nonhuman viral DNA) within the human genome. During the next five years we will focus on a fundamental understanding of the thermodynamics of oligonucleotide-directed triple helix formation, initiate high resolution structure studies, study mechanistic details of quantitative covalent reactions within a triple helix complex such as alkylation and hydrolysis, synthesize novel ligands for simultaneous major/minor groove binding, and develop methods and strategies for in vivo studies of triple helix formation. Specifically, we will optimize and compare methods such as quantitative affinity cleaving titration and quantitative DNase I footprint titrations for characterizing the equilibrium constants and free energies values for association of oligonucleotides for single sites on relatively large double helical DNA under a broad range of solution conditions. We will examine the energetics of all 16 natural triplets in the pyrimidine.purine.pyrimidine triple helix motif and all 16 natural triplets in the purine.pyrimidine.purine triple helix motif. We will quantitate the temperature dependence and nearest neighbor dependence of triple helix formation. We will quantitate the energetics of triple helix stabilities with regard to base modifications (5-1-propynyl, PI), sugar modifications (2'-OH, -OMe, O-allyl) and phosphodiester modifications (stereoregular phosphorothioate). We will make a major commitment toward structural characterization of triple helical complexes by x-ray crystallography. We will carry out a kinetic analysis of sequence specific alkylation of double helical DNA by an N-bromoacetyloligonucleotide. We will extend these methods to sequence specific double strand cleavage of RNA. We will synthesize novel catalysts for the sequence specific hydrolysis of DNA and RNA. We will design and synthesize novel peptide-oligonucleotide hybrids for simultaneous cooperative binding in the minor and major groove of DNA. In a collaboration, we will combine our chemical strengths with an expert biology group to study triple helix formation in vivo.